Designing potential HDAC3 inhibitors to improve memory and learning

The work presented here explores the structural and physicochemical features important for benzamide-based HDAC3 inhibitors to get an idea about the design aspect of potential inhibitors. A number of molecular modeling studies (3D-QSAR CoMFA and CoMSIA, Bayesian classification modeling) were performed on 113 diverse set of benzamide-based HDAC3 inhibitors. All these models developed are statistically reliable and correlate the SAR observations. Electron withdrawing substitution is favorable but the bulky hydrophobic group at the cap region reduces HDAC3 inhibition. Hydrophobicity and steric feature of the aryl linker function favor the activity. Aryl group substituted benzamide functionality is not favorable for HDAC3 inhibition. The amide function of the benzamide moiety is essential for Zn²⁺ chelation and the carboxylic acid function may serve as a hydrogen bond acceptor (HBA) feature. Moreover, electron withdrawing substituent at the benzamide moiety influences activity whereas steric and hydrophobic substituents reduce HDAC3 inhibition. Overall, this study may provide a valuable insight on the design of better active HDAC3 inhibitors in future.

Communicated by Ramaswamy H. Sarma     

Combined pharmacophore modeling, 3D-QSAR and docking studies to identify novel HDAC inhibitors using drug repurposing

Abstract

Histone deacetylases (HDACs), a critical family of epigenetic enzymes, has emerged as a promising target for antitumor drugs. Here, we describe our protocol of virtual screening in identification of novel potential HDAC inhibitors through pharmacophore modeling, 3D-QSAR, molecular docking and molecular dynamics (MD) simulation. Considering the limitation of current virtual screening works, drug repurposing strategy was applied to discover druggable HDAC inhibitor. The ligand-based pharmacophore and 3D-QSAR models were established, and their reliability was validated by different methods. Then, the DrugBank database was screened, followed by molecular docking. MD simulation (100?ns) was performed to further study the stability of ligand binding modes. Finally, results indicated the hit DB03889 with high in silico inhibitory potency was suitable for further experimental analysis.

Communicated by Ramaswamy H. Sarma     

HDAC8 drives spindle organization during meiotic maturation of porcine oocytes

ObjectivesHistone deacetylase 8 (HDAC8) is one of the class I HDAC family proteins, which participates in the neuronal disorders, parasitic/viral infections, tumorigenesis and many other biological processes. However, its potential function during female germ cell development has not yet been fully understood.Materials and methodsHDAC8‐targeting siRNA was microinjected into GV oocytes to deplete HDAC8. PCI‐34051 was used to inhibit the enzyme activity of HDAC8. Immunostaining, immunoblotting and fluorescence intensity quantification were applied to assess the effects of HDAC8 depletion or inhibition on the oocyte meiotic maturation, spindle/chromosome structure, γ‐tubulin dynamics and acetylation level of α‐tubulin.ResultsWe observed that HDAC8 was localized in the nucleus at GV stage and then translocated to the spindle apparatus from GVBD to M II stages in porcine oocytes. Depletion of HDAC8 led to the oocyte meiotic failure by showing the reduced polar body extrusion rate. In addition, depletion of HDAC8 resulted in aberrant spindle morphologies and misaligned chromosomes due to the defective recruitment of γ‐tubulin to the spindle poles. Notably, these meiotic defects were photocopied by inhibition of HDAC8 activity using its specific inhibitor PCI‐34051. However, inhibition of HDAC8 did not affect microtubule stability as assessed by the acetylation level of α‐tubulin.ConclusionsCollectively, our findings demonstrate that HDAC8 acts as a regulator of spindle assembly during porcine oocyte meiotic maturation.     

Discovery of a series of hydroximic acid derivatives as potent histone deacetylase inhibitors

Abstract

To develop potent histone deacetylase inhibitors as antitumor agents, structural modification was performed. The synthesized molecules were tested by enzymatic inhibition assay and anti-proliferation assay. Several molecules show improved activities in the enzymatic inhibition assay. However, in the MTT assays, all these derived molecules have limited performance compared with SAHA. The IC50 values of molecule ((S)-N-(6-(hydroxyamino)-6-oxohexyl)-4-(3-(2-oxo-1-phenyl-2-((3-(trifluoromethyl)phenyl)amino)ethyl)ureido)benzamide, L8) which has the best enzymatic inhibition activity (with an IC50 value of 11.7?nm and 967?nm against Hela nucleus extract and HDAC8, respectively) were calculated compared with SAHA. Molecular docking was performed to predict the binding mode of molecule L8 in the active site of HDAC2 and HDAC8. Hydrophobic interaction, chelate binding, electrostatic attraction and H-bond interaction in combination make contribution to the ligand–receptor interactions.     

Potent non-hydroxamate inhibitors of histone deacetylase-8: Role and scope of an isoindolin-2-yl linker with an α-amino amide as the zinc-binding unit

A series of potent inhibitors of histone deacetylase-8 (HDAC8) is described that contains an α-amino amide zinc-binding unit and a substituted isoindolinyl capping group. The presence of a 2,4-dichlorophenyl unit located in the acetate-release cavity was shown to confer a gain of approx. 4.3 kJ mol⁻¹ in binding energy compared to a phenyl group, and the isoindoline linker has approx. 5.8 kJ mol⁻¹ greater binding energy than the corresponding tetrahydroisoquinoline ring system. In a series of 5-substituted isoindolin-2-yl inhibitors, a 5-acetylamino derivative was found to be more potent than the 5-unsubstituted lead HDAC8 inhibitor (increase in binding energy of 2.0 kJ mol⁻¹, ascribed to additional binding interactions within the N_ε-acetyl-l-lysine binding tunnel in HDAC8, including hydrogen bonding to Asp101. Tolerance of a 5-substituent (capping group) on the isoindoline ring has been demonstrated, and which in some cases confers improved enzyme inhibition, the HDAC8 substrate-binding region providing a platform for additional interactions.     

Novel histone deacetylase 8 ligands without a zinc chelating group: Exploring an ‘upside-down’ binding pose

A novel series of HDAC8 inhibitors without a zinc-chelating hydroxamic acid moiety is reported. Photoaffinity labeling and molecular modeling studies suggest that these ligands are likely to bind in an ‘upside-down’ fashion in a secondary binding site proximal to the main catalytic site. The most potent ligand in the series exhibits an IC₅₀ of 28 μM for HDAC8 and is found to inhibit the deacetylation of H4 but not α-tubulin in SH-SY5Y cell line.     

Irreversible,Tight-Binding Inhibition of Adenosine Deaminase by Coformycins: Inhibitor Structural Features That Contribute to the Mode of Enzyme Inhibition

Abstract

Coformycin analogues 1–6 were synthesized and biochemically screened against adenosine deaminase in order to assess the relative contributions of N-4, N-6, and the N-3 sugar moiety to the mode of enzyme inhibition. Our results indicate that N-4 plays a relatively greater role than N-6 in enzyme tight-binding, and that a benzyl group can substitute for the sugar moiety at N-3. The absence of a sugar or benzyl group at N-3, however, leads to loss of activity. The hydroxyl group at C-8, while crucial for activity, does not alone confer the tight-binding characteristics to coformycins.     

Evaluation of functional groups on amino acids in cyclic tetrapeptides in histone deacetylase inhibition

The naturally occurring cyclic tetrapeptide, chlamydocin, originally isolated from fungus Diheterospora chlamydosphoria, consists of α-aminoisobutyric acid, l-phenylalanine, d-proline and an unusual amino acid (S)-2-amino-8-((S)-oxiran-2-yl)-8-oxooctanoic acid (Aoe) and inhibits the histone deacetylases (HDACs), a class of regulatory enzymes. The epoxyketone moiety of Aoe is the key functional group for inhibition. The cyclic tetrapeptide scaffold is supposed to play important role for effective binding to the surface of enzymes. In place of the epoxyketone group, hydroxamic acid and sulfhydryl group have been applied to design inhibitor ligands to zinc atom in catalytic site of HDACs. In the research for more potent HDAC inhibitors, we replaced the epoxyketone moiety of Aoe with different functional groups and synthesized a series of chlamydocin analogs as HDAC inhibitors. Among the functional groups, methoxymethylketone moiety showed as potent inhibition as the hydroxamic acid. On the contrary, we confirmed that borate, trifruoromethylketone, and 2-aminoanilide are almost inactive in HDAC inhibition.     

Structural Basis for the Inhibition of Histone Deacetylase 8 (HDAC8), a Key Epigenetic Player in the Blood Fluke Schistosoma mansoni

The treatment of schistosomiasis, a disease caused by blood flukes parasites of the Schistosoma genus, depends on the intensive use of a single drug, praziquantel, which increases the likelihood of the development of drug-resistant parasite strains and renders the search for new drugs a strategic priority. Currently, inhibitors of human epigenetic enzymes are actively investigated as novel anti-cancer drugs and have the potential to be used as new anti-parasitic agents. Here, we report that Schistosoma mansoni histone deacetylase 8 (smHDAC8), the most expressed class I HDAC isotype in this organism, is a functional acetyl-L-lysine deacetylase that plays an important role in parasite infectivity. The crystal structure of smHDAC8 shows that this enzyme adopts a canonical α/β HDAC fold, with specific solvent exposed loops corresponding to insertions in the schistosome HDAC8 sequence. Importantly, structures of smHDAC8 in complex with generic HDAC inhibitors revealed specific structural changes in the smHDAC8 active site that cannot be accommodated by human HDACs. Using a structure-based approach, we identified several small-molecule inhibitors that build on these specificities. These molecules exhibit an inhibitory effect on smHDAC8 but show reduced affinity for human HDACs. Crucially, we show that a newly identified smHDAC8 inhibitor has the capacity to induce apoptosis and mortality in schistosomes. Taken together, our biological and structural findings define the framework for the rational design of small-molecule inhibitors specifically interfering with schistosome epigenetic mechanisms, and further support an anti-parasitic epigenome targeting strategy to treat neglected diseases caused by eukaryotic pathogens.     

The discovery of novel HDAC3 inhibitors via virtual screening and in vitro bioassay

Histone deacetylase 3 (HDAC3) is a potential target for the treatment of human diseases such as cancers, diabetes, chronic inflammation and neurodegenerative diseases. Previously, we proposed a virtual screening (VS) pipeline named “Hypo1_FRED_SAHA-3” for the discovery of HDAC3 inhibitors (HDAC3Is) and had thoroughly validated it by theoretical calculations. In this study, we attempted to explore its practical utility in a large-scale VS campaign. To this end, we used the VS pipeline to hierarchically screen the Specs chemical library. In order to facilitate compound cherry-picking, we then developed a knowledge-based pose filter (PF) by using our in-house quantitative structure activity relationship- (QSAR-) modelling approach and coupled it with FRED and Autodock Vina. Afterward, we purchased and tested 11 diverse compounds for their HDAC3 inhibitory activity in vitro. The bioassay has identified compound 2 (Specs ID: AN-979/41971160) as a HDAC3I (IC₅₀?=?6.1?μM), which proved the efficacy of our workflow. As a medicinal chemistry study, we performed a follow-up substructure search and identified two more hit compounds of the same chemical type, i.e. 2–1 (AQ-390/42122119, IC₅₀?=?1.3?μM) and 2–2 (AN-329/43450111, IC₅₀?=?12.5?μM). Based on the chemical structures and activities, we have demonstrated the essential role of the capping group in maintaining the activity for this class of HDAC3Is. In addition, we tested the hit compounds for their in vitro activities on other HDACs, including HDAC1, HDAC2, HDAC8, HDAC4 and HDAC6. We have identified these compounds are HDAC1/2/3 selective inhibitors, of which compound 2 show the best selectivity profile. Taken together, the present study is an experimental validation and an update to our earlier VS strategy. The identified hits could be used as starting structures for the development of highly potent and selective HDAC3Is.     

Novel Histone Deacetylase Class IIa Selective Substrate Radiotracers for PET Imaging of Epigenetic Regulation in the Brain

Histone deacetylases (HDAC’s) became increasingly important targets for therapy of various diseases, resulting in a pressing need to develop HDAC class- and isoform-selective inhibitors. Class IIa deacetylases possess only minimal deacetylase activity against acetylated histones, but have several other client proteins as substrates through which they participate in epigenetic regulation. Herein, we report the radiosyntheses of the second generation of HDAC class IIa–specific radiotracers: 6-(di-fluoroacetamido)-1-hexanoicanilide (DFAHA) and 6-(tri-fluoroacetamido)-1-hexanoicanilide ([¹⁸F]-TFAHA). The selectivity of these radiotracer substrates to HDAC class IIa enzymes was assessed in vitro, in a panel of recombinant HDACs, and in vivo using PET/CT imaging in rats. [¹⁸F]TFAHA showed significantly higher selectivity for HDAC class IIa enzymes, as compared to [¹⁸F]DFAHA and previously reported [¹⁸F]FAHA. PET imaging with [¹⁸F]TFAHA can be used to visualize and quantify spatial distribution and magnitude of HDAC class IIa expression-activity in different organs and tissues in vivo. Furthermore, PET imaging with [¹⁸F]TFAHA may advance the understanding of HDACs class IIa mediated epigenetic regulation of normal and pathophysiological processes, and facilitate the development of novel HDAC class IIa-specific inhibitors for therapy of different diseases.     

Investigation of Decitabine Effects on HDAC3 and HDAC7 mRNA Expression in NALM-6 and HL-60 Cancer Cell Lines

Background:Decitabine is a potent anticancer hypomethylating agent and changes the gene expression through the gene''s promoter demethylation and also independently from DNA demethylation. So, the present study was designed to distinguish whether Decitabine, in addition to inhibitory effects on DNA methyltransferase, can change HDAC3 and HDAC7 mRNA expression in NALM-6 and HL-60 cancer cell lines.Methods:HL-60, NALM-6, and normal cells were cultured, and the Decitabine treatment dose was obtained (1 µM) through the MTT assay. Finally, HDAC3 and HDAC7 mRNA expression were measured by Real-Time PCR in HL-60 and NALM-6 cancerous cells before and after treatment. Furthermore, HDAC3 and HDAC7 mRNA expression in untreated HL-60 and NALM-6 cancerous cells were compared to normal cells.Results:Our results revealed that the expression of HDAC3 and HDAC7 in HL-60 and NALM-6 cells increases as compared to normal cells. After treatment of HL-60 and NALM-6 cells with Decitabine, HDAC3, and HDAC7 mRNA expression were decreased significantly.Conclusion:Our data confirmed that the effects of Decitabine are not limited to direct hypomethylation of DNMTs, but it can indirectly affect other epigenetic factors, such as HDACs activity, through converging pathways.Key Words: Decitabine, HDAC3, HDAC7, HL-60, NALM-6     

Class I HDAC imaging using [3H]CI-994 autoradiography

[³H]CI-994, a radioactive isotopologue of the benzamide CI-994, a class I histone deacetylase inhibitor (HDACi), was evaluated as an autoradiography probe for ex vivo labeling and localizing of class I HDAC (isoforms 1–3) in the rodent brain. After protocol optimization, up to 80% of total binding was attributed to specific binding. Notably, like other benzamide HDACi, [³H]CI-994 exhibits slow binding kinetics when measured in vitro with isolated enzymes and ex vivo when used for autoradiographic mapping of HDAC1–3 density. The regional distribution and density of HDAC1–3 was determined through a series of saturation and kinetics experiments. The binding properties of [³H]CI-994 to HDAC1–3 were characterized and the data were used to determine the regional B_max of the target proteins. K_d values, determined from slice autoradiography, were between 9.17 and 15.6 nM. The HDAC1–3 density (B_max), averaged over whole brain sections, was of 12.9 picomol · mg⁻¹ protein. The highest HDAC1–3 density was found in the cerebellum, followed by hippocampus and cortex. Moderate to low receptor density was found in striatum, hypothalamus and thalamus. These data were correlated with semi-quantitative measures of each HDAC isoform using western blot analysis and it was determined that autoradiographic images most likely represent the sum of HDAC1, HDAC2, and HDAC3 protein density. In competition experiments, [³H]CI-994 binding can be dose-dependently blocked with other HDAC inhibitors, including suberoylanilide hydroxamic acid (SAHA). In summary, we have developed the first known autoradiography tool for imaging class I HDAC enzymes. Although validated in the CNS, [³H]CI-994 will be applicable and beneficial to other target tissues and can be used to evaluate HDAC inhibition in tissues for novel therapies being developed. [³H]CI-994 is now an enabling imaging tool to study the relationship between diseases and epigenetic regulation.     

Molecular Modeling Study on Tunnel Behavior in Different Histone Deacetylase Isoforms

Histone deacetylases (HDACs) have emerged as effective therapeutic targets in the treatment of various diseases including cancers as these enzymes directly involved in the epigenetic regulation of genes. However the development of isoform-selective HDAC inhibitors has been a challenge till date since all HDAC enzymes possess conserved tunnel-like active site. In this study, using molecular dynamics simulation we have analyzed the behavior of tunnels present in HDAC8, 10, and 11 enzymes of class I, II, and IV, respectively. We have identified the equivalent tunnel forming amino acids in these three isoforms and found that they are very much conserved with subtle differences to be utilized in selective inhibitor development. One amino acid, methionine of HDAC8, among six tunnel forming residues is different in isoforms of other classes (glutamic acid (E) in HDAC10 and leucine (L) in HDAC 11) based on which mutations were introduced in HDAC11, the less studied HDAC isoform, to observe the effects of this change. The HDAC8-like (L268M) mutation in the tunnel forming residues has almost maintained the deep and narrow tunnel as present in HDAC8 whereas HDAC10-like (L268E) mutation has changed the tunnel wider and shallow as observed in HDAC10. These results explained the importance of the single change in the tunnel formation in different isoforms. The observations from this study can be utilized in the development of isoform-selective HDAC inhibitors.     

Discovery of a series of small molecules as potent histone deacetylase inhibitors

Abstract

A series of small molecules were designed and synthesized based on our previous virtual screening approach, which was performed to discover potent histone deacetylase inhibitors (HDACIs) with novel structures. The derived compounds were tested by Hela cell nucleus extract for enzyme inhibition assay. Tumor cell growth inhibition assays were performed using a series of tumor cell lines. Molecule 4h has the best performance among these compounds with enzyme inhibition IC₅₀ of 0.14?μM and tumor cell growth inhibition IC₅₀ of 1.85 (U937), 2.02 (HL60), 2.67 (K562). Docking studies showed that multiple H-bonds and hydrophobic interactions make 4h binding to the active site of HDAC. 4h has the advantage of low molecular weight, so a variety of structural modifications can be performed in our further studies.     

Histone deacetylase 1 and 2 differentially regulate apoptosis by opposing effects on extracellular signal-regulated kinase 1/2

Histone deacetylases (HDACs) are epigenetic regulators that are important for the control of various pathophysiological events. We found that HDAC inhibitors completely abolished transforming growth factor-β1 (TGF-β1)-induced apoptosis in AML-12 and primary mouse hepatocytes. Expression of a dominant-negative mutant of HDAC1 or downregulation of HDAC1 by RNAi both suppressed TGF-β1-induced apoptosis. In addition, overexpression of HDAC1 enhanced TGF-β1-induced apoptosis, and the rescue of HDAC1 expression in HDAC1 RNAi cells restored the apoptotic response of cells to TGF-β1. These data indicate that HDAC1 functions as a proapoptotic factor in TGF-β1-induced apoptosis. In contrast, downregulation of HDAC2 by RNAi increased spontaneous apoptosis and markedly enhanced TGF-β1-induced apoptosis, suggesting that HDAC2 has a reciprocal role in controlling cell survival. Furthermore, inhibition of extracellular signal-regulated kinase 1/2 (ERK1/2) by MEK1 inhibitor PD98059 or expression of a kinase-dead mutant of MEK1 restored the apoptotic response to TGF-β1 in HDAC1 RNAi cells. Strikingly, HDAC2 RNAi caused an inhibition of ERK1/2, and the spontaneous apoptosis can be abolished by reactivation of ERK1/2. Taken together, our data demonstrate that HDAC1 and 2 reciprocally affect cell viability by differential regulation of ERK1/2; these observations provide insight into the roles and potential mechanisms of HDAC1 and 2 in apoptosis.